Multiphoton microscopy is a technique that allows imaging of thick, living tissue with submicron spatial resolution. We have developed and used this technique to ask questions about the progression and prevention of amyloid pathology in mouse models of Alzheimer's disease. Using a variety of both structural and functional fluorophores, we have developed ways to image morphology and activity in specific cell types in the living brain. Using fluorescence lifetime imaging microscopy (FLIM), we have elucidated the interactions of a broad spectrum of proteins implicated in neurodegenerative diseases as well as in other tissues and organs by applying FLIM to measures of fluorescence resonance energy transfer (FRET). The aims of this proposal are to expand on this progress and image deeper, image with higher axial resolution, and push lifetime imaging further with multispectral FLIM approaches. These technological advances to improve multiphoton imaging techniques will be applied to structural and functional readouts of the principle cell types (neurons, astrocytes, and microglia) in the brains of living mice. Our biological applications will focus on mouse models of Alzheimer's disease, but the techniques will be broadly applicable to many other health and disease models, and not restricted just to the brain. Aim 1 will use multispectral FLIM for high content imaging. We will develop analytical approaches to exploit the interaction of fluorescence decays with spectra. These developments will allow better, more robust FRET measurements and removal of complicated autofluorescence signatures. Aim 2 will implement a pulsed laser source operating in the 1-1.6 micron spectral range for very deep 2-photon imaging of far red and near infrared fluorophores. We will evaluate the depth of imaging with this new approach and expect to achieve structural imaging of neurons up to 1mm into the brain. This source will also allow much improved axial resolution with 3-photon excitation of visible light fluorophores. Aim 3 will apply these technological advances to structural and functional cellular imaging in the living brain. We will capitalize on existing and emerging reporters to monitor cellular structure deep in the living brain and measure functional activity in response to local and sensory stimulation to probe neuronal and astroglial network activity. All together, these technological advances will support in vivo cellular imaging as a platform to understand brain function and treat neurological diseases.